Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease characterized by chronic hemolysis causing hemoglobinuria, bone marrow (BMF) failure, and a tendency to thrombosis. PNH is due to the expansion of an abnormal done which arises through a somatic mutation in the X- linked PIG-A gene on the active X-chromosome in an early hematopoietic stem cell. The PIG-A mutation abolishes or impairs the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Cells belonging to the FNH done are therefore deficient in all proteins that are anchored to the membrane through GPI anchors. PNH is closely related to aplastic anemia (AA), in that the incidence of PNH is increased in patients with AA, and AA is often a dreaded end-stage of PNH. This proposal aims to test the working hypothesis that: (a) PNH is always associated with some degree of BMF; (b) hematopoietic clones with the PNH phenotype (GPI-negative) have a survival and/or growth advantage under conditions that cause or maintain BMF. To test this hypothesis the plan is to create an animal model, in which some or all of hematopoiesis lacks all GPI-linked proteins. For this purpose inactivation of the PIG-A gene in mouse embryonic stem (ES) cells has been already achieved. These 'PIG-A-null' ES cells will now be used in order to study their differentiation in-vitro, and their ability to contribute to mouse development after transfer into blastocysts. Since the phenotype of a fully PIG-A null animal might be severe or not compatible with life, several different approaches will be explored to achieve viable PNH hematopoiesis. (1) Rescue of mutants with impaired hematopoiesis (e.g. W/W) by transfer of PIG-A null ES cells into mutant blastocysts. (2) Transplantation of hematopoietic cells derived from PIG-A-null ES-cell into irradiated recipients. Finally, PIG-A positive and PIG-A null hematopoiesis will be compared within the same animal under normal conditions, as well as under conditions that cause or maintain BMF.